Grantee Research Project Results

Final Report: Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition

EPA Grant Number: R832415C005
Subproject: this is subproject number 005 , established and managed by the Center Director under grant R832415
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Rochester PM Center
Center Director: Oberdörster, Günter
Title: Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition
Investigators: Finkelstein, Jacob N. , Prather, Kimberly A. , Rahman, Arshad , Oakes, David , Phipps, Richard
Institution: University of Rochester , University of California - San Diego
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2012)
RFA: Particulate Matter Research Centers (2004) RFA Text | Recipients Lists
Research Category: Human Health , Air

Objective:

Experiments in this core were designed to provide a mechanistic link and biological plausibility for the whole animal and controlled clinical (human) exposures. Our ability to use defined populations of cells and well characterized particles allows to test specific hypothesis that arise from the in vivo studies described elsewhere. Endothelial cells as a potential target for translocated ultrafine particles were used to compare different effects of several particulate materials with those of ambient concentrated particles. Results showed that induction of IL-6 gene expression and production of nitrate is regulated by independent mechanisms and simply invoking particle-induced oxidative stress is insufficient to our understanding of the process.

Summary/Accomplishments (Outputs/Outcomes):

A particular focus of the in vitro studies was to identify mechanisms that may be involved in the enhanced susceptibility of cells from diabetics. Our in vitro studies were designed to model this under controlled conditions. One of the hallmarks of the diabetic is the increased blood glucose and we have shown that culture of vascular endothelial cells in high glucose alters both the basal and particle induced cytokine responses. Using this model we studied the response of pulmonary cells to particles collected by a high volume sampler in Rochester. These are similar to PM used in animal and human clinical studies carried out using the Harvard ultrafine particle concentrator. Methods: Human respiratory epithelial cells exposed to collected ultrafine particles responded through increased production of IL-6. Results: In contrast to previous work that focused only on the production of NO and endothelium epithelial cells maintained under conditions of hyperglycemia actually produced increased amounts of IL-6. This was in contrast to results where HUVEC were cultured under similar conditions. It was also evident that the ambient samples showed a somewhat different response in these cells for this marker.

Title: Molecular mechanisms leading to altered gene expression in relation to particle composition

Emphasis in these studies shifted to vascular cells to examine specific mechanistic pathways that complement results of the cardiovascular in vivo endpoints. Endothelial cell monolayer as well as epithelial-endothelial cell co-cultures were established. Exposures to different ultrafine particle types or " as positive control LPS "showed that measurement of induced IL-6 has best predictive value. The co-culture model used the epithelial A549 cell on the top of a transwell insert with HUVEC cells in the lower chamber. Adding a stimulus to the upper chamber stimulated directly the epithelial cells, while dosing the lower chamber stimulated the apical side of the endothelial cells. Results with ultrafine particles show specificity in terms of oxidative stress induction observed only after particle addition to the upper but not to the lower chamber. Use of collected ambient ultrafine particles for in vitro cell culture studies did experience problems in that it was not possible to separate the particles from the filter substrate.

Title: Mechanisms of the interactions between ultrafine particles from different sources and specific pulmonary cell populations related to oxidative stress induction

Results: We have focused on developing approaches that could be used to measure cellular responses to collected and fractionated sample of ambient ultrafine PM. PM samples obtained from various sources were tested in a novel growth tube device that would increase the yield of ultrafine PM material available for testing. Each sample was adjusted to the same final concentration and evaluated in our indicator A549 cell line. Physical and chemical ambient measurements of the samples collected between the ports of Long Beach and Los Angeles showed that the major sources of the particles were marine diesel combustion, local diesel pollution, nearby freeway pollution, biomass burning, and transport of pollution from California's Central Valley. The results indicate that ambient aerosol containing ultrafine PM from marine diesel engines are significantly more reactive inducing oxidative stress than ultrafine TiO2 particles that were used as control PM. However, the yield of the growth tube device was insufficient for obtaining enough ultrafine PM sample to establish dose-response relationships. Efforts to increase the yield of the collecting system were not successful. We were experiencing problems with collecting source-specific ambient ultrafine particles using a new growth tube/impinger system for studying potential responses in vitro. A larger model of the growth tube had been developed to be used for collecting source-specific ultrafine particles but was still not able to supply sufficient ultrafine PM material to carry out meaningful in vitro studies.

Conclusions:

In vitro studies were designed to model the diabetic condition and thereby altered cellular responses to PM by culturing vascular endothelial cells in high glucose. This altered both the basal and particle-induced cytokine response. We showed that the normally increased production of NO in ultrafine particle exposed endothelial cells is reduced under these hyperglycemic culture conditions. We found also that particulate matter from different sites show significant differences in their ability to induce NO production, suggesting to reflect differences in PM composition.

One question raised in our studies is the role of PM induced oxidative stress in the generation of cytokine or NO ( Nitric Oxide) responses. The human clinical studies have been measuring vascular reactivity as a measure of response to inhaled PM. Included in that battery of outcomes was IL-6 and plasma Nitric Oxide (NO). To examine this response in a mechanistic manner we measured changes in both of these outcomes in cells that have had their antioxidant status altered by culturing with exogenous antioxidants.

Conclusions from the in vitro studies are:

Elevated glucose, as a model for diabetes, caused enhanced cytokine production by endothelial cells when cultured with collected ambient samples of PM.
No production was significantly affected by PM composition and ongoing oxidative stress in cultured endothelial cells.
Modeling endothelial cell responses to PM was enhanced in a co-culture model where both epithelial cells and endothelial cells were exposed to PM.
Source-specific PM can be used in vitro as a potential model to examine the role of specific PM components.
Marine diesel PM showed the greatest oxidative stress potential in vitro.

Journal Articles:

No journal articles submitted with this report: View all 13 publications for this subproject

Supplemental Keywords:

Health, RFA, Scientific Discipline, Air, PHYSICAL ASPECTS, Health Risk Assessment, Physical Processes, Risk Assessments, particulate matter, Biology, Genetics, altered gene expression, human exposure, long term exposure, aersol particles, atmospheric particles, ambient particle health effects, exposure, atmospheric aerosol particles, PM, atmospheric particulate matter, human health risk

Progress and Final Reports:

Original Abstract

Main Center Abstract and Reports:

R832415 Rochester PM Center

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R832415C001 Characterization and Source Apportionment
R832415C002 Epidemiological Studies on Extra Pulmonary Effects of Fresh and Aged Urban Aerosols from Different Sources
R832415C003 Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles
R832415C004 Animal models: Cardiovascular Disease, CNS Injury and Ultrafine Particle Biokinetics
R832415C005 Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.